Evaluation of a rapid fractionation procedure of rabbit reticulocytes after digitonin or hypotonic hemolysis for the study of enzyme and metabolite distributions.
A rapid centrifugation procedure following cell lysis with either digitonin or short hypotonic shock in EDTA containing solutions was evaluated, applicable for investigations of enzyme and metabolite compartmentations in intact rabbit reticulocytes. The application of the digitonin disruption seems to be restricted to cell suspensions containing up to 40% reticulocytes only, whereas the hypotonic lysis can be used with practically pure reticulocytes, too. The distribution of markers revealed that an almost complete cell disruption, sufficient separation into pellet and supernatant fraction and satisfactory preservation of mitochondrial intactness could be achieved under appropriate conditions. The suitability of the proposed method in studies on reticulocyte energy metabolism is further supported by the almost insignificant ATP splitting during the entire procedure.
D000255:Adenosine Triphosphate / Q000097:blood; D000740:Anemia / Q000097:blood; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D004072:Digitonin / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000097:blood; D006461:Hemolysis; D007038:Hypotonic Solutions; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism
D007038:Hypotonic Solutions; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D005969:Glutamate Dehydrogenase; D004072:Digitonin